Fluorescent Molecular Rotors
Molecular rotors are a collective group of fluorescent compounds that possess the ability to undergo twisted intramolecular charge transfer (TICT) and are typically used as viscosity sensor probes. They typically consist of three parts: an electron-donating unit, an electron-accepting unit and a π-conjugated linking moiety which allows electron transfer to occur in the planar conformation. However, electrostatic forces upon irradiation result in the molecule adopting a twisted conformation around the σ-bond in the linker region. This non-planar, twisted conformation has a lower excited state energy and thus is associated with either a red-shifted fluorescence emission or can undergo a non-radiative torsional relaxation pathway, depending on the molecular structure of the rotor. If the intramolecular rotation is hindered, the non-radiative pathway is prevented and the molecule adopts a planar configuration, thus restoring fluorescence.
Protein Labelling Techniques
Progress in understanding complex biological systems depends on characterizing the underlying interactions of biomolecules, in particular proteins. The interaction of proteins with ligands, such as peptides, DNA or small molecules, offers biologists powerful tools for visualizing protein dynamics.
For example, the investigation of protein-peptide interactions. The p53 tumour suppressor protein is the key determinant of cell fate. It is mutated in 50% of all cancers. It is primarily regulated by the ubiquitin ligase MDM2 which targets it for proteosomal degradation. The interaction between MDM2 and p53 has been mapped to the N-terminal of p53 (residues 18-26) and the N-terminal domain of MDM2 (residues 1-110). In 50% of cancers with wild-type p53, inhibition of MDM2 leading to increased p53 levels and cell death represents an attractive therapeutic modality. Several compounds that bind to the N-terminal domain of MDM2 and abrogate p53 binding have shown promise in preclinical development. Both the further development of these pre-existing compounds and high-throughput screens for novel compounds will benefit greatly from robust, facile and sensitive methods enabling detection of the p53-MDM2 interaction.
Additionally, the interactions of proteins with DNA are essential cellular processes. Compromised protein-DNA interactions can give rise to severe disease phenotypes, notably cancer. There exists therefore, a requirement for robust assays enabling both fundamental understanding of interactions at the molecular level, and high-throughput screening of compound libraries for drugs capable of “reactivating” a mutant protein with diminished or absent DNA-binding.
One way to determine protein-DNA binding is through electrophoretic mobility shift assay (EMSA). EMSA identifies protein-DNA binding by the shift in the electrophoretic migration of DNA through a gel when it is bound by a protein. Another way is through and DNA footprinting. DNA footprinting identifies protein-DNA complexes through resistance of DNA to nucleolytic degradation when it is bound by a protein. However, these methods are technically demanding, semi-quantitative, not-easily reproduced, low-throughput and typically require the use of radioisotopes for optimal results.
Another way is based on the ELISA format involving the use of biotinylated DNA to capture protein-DNA (p53 protein bound to target DNA) complexes on streptavidin plates. The complexes are subsequently detected through the use of an anti-p53 monoclonal antibody that does do not disrupt the complex. A variation of this technique has also been described using microspheres and flow analysis. Other techniques include surface plasmon resonance (SPR), and fluorescence anisotropy. Whilst powerful and insightful, these methods require expensive instrumentation, are laborious, and are not suited for high-throughput applications.
One way of detecting protein-DNA binding occurs through a combination of immunoprecipitation and real-time PCR. However, this method is not optimal for high-throughput screening as it requires multiple washing steps and real-time PCR which can be costly.
There is therefore a need to provide a quantitative, label-free, homogenous, non-radioactive, reproducible and high-throughput method to measure protein-ligand binding.
There is also a need to provide a high-throughput screening method for measuring protein-ligand interactions which is non-laborious and does not require the use of expensive instrumentation.
There is also a need to provide a high-throughput screening method for measuring protein-ligand interactions which lessens the requirement for multiple washing steps.
Therefore, there is a need for methods for detecting interactions between a protein and ligands that ameliorate the above problems. The present invention seeks to fulfill these needs and provides further related advantages.